The proposed research will be a combined experimental and computational study of the interactions between protein kinases and small molecule inhibitors. Protein kinases are implicated in a number of diseases, and are therefore attractive therapeutic targets. A major challenge however in designing small molecule inhibitors of kinases is how to develop inhibitors that specifically target a kinase of interest while leaving the hundreds of other kinases in the body unaffected. In order to achieve this goal, the factors that control the specificity of inhibitor-kinase interactions need to be understood, and one way to do this is to develop computational methodologies that can accurately predict the relative affinities of inhibitor-kinase complexes. The proposed research plan aims to test, extend and refine such a computational methodology through combination with a number of experimental studies. The specific aims involve: (1) Inhibition constants of commercially available small molecule inhibitors with a range of approximately 10 protein kinase targets will be measured. These studies will provide new quantitative data on inhibitor-kinase interactions. (2) The binding thermodynamics of inhibitor-kinase complexes will be measured with isothermal titration calorimetry. Separate measurements of binding free energies, enthalpies and entropies will be used to parameterize an energetic description of inhibitor-kinase binding interactions. (3) The crystal structures of inhibitors in complex with a range of different protein kinase targets will be solved. These studies will provide a direct and comprehensive view of the extent to which the binding orientations adopted by a given inhibitor change when complexed with different protein kinases. (4) The experimental data obtained from Aims 1-3 will be used to develop and test a computational method for accurately predicting the relative binding affinities of an inhibitor with all approximately 500 human protein kinases. Protein kinases, being implicated in a large number of different cancer types, are increasingly seen as very important targets for therapeutic drugs. The research proposed here directly addresses the single greatest challenge to the therapeutic pursuit of kinases: how to design small molecule inhibitors that can specifically inhibit a kinase of interest.